PROJECT SUMMARY Astrocytic gliomas are the most common type of malignant brain tumor. Even with treatment, the average life expectancy for the most malignant grade is only 15 months. High grade gliomas are especially complicated to diagnose and treat due to their infiltrative nature and low accessibility (i.e., blood brain-barrier, skull). By the time it is realized, the tumor is quite advanced. The standard of care is surgical removal of the tumor followed by chemo/radiotherapy, with the most conclusive prognostic factor being extent of removal. A greater understanding of the molecular landscape is necessary in order to develop more effective and personalized treatments. In addition, targeted high-contrast agents would find use for surgical resections where exact removal of all and only cancerous tissue is vital. Researchers have identified numerous biomarkers that can differentiate cancerous from healthy tissues and serve as prognostic markers. If incorporated into an activatable probe, these biomarkers could be used for fluorescence-guided surgery to make visualization of cancerous tissues more evident. That way, the surgeon is more apt to remove all the cancer which increases lifespan and reduces remission rates. One such biomarker is cathepsin B, a lysosomal cysteine protease that is involved in cellular protein turnover, overexpressed in highly malignant brain gliomas, and shown to be involved in tumor invasion and migration. Therefore, we aim to synthesize a novel molecular probe to image cathepsin B activity in astrocytic gliomas. The probe has several key components: a fluorophore, a radioactive positron emitter, a peptide vector that allows it to cross the blood-brain barrier, and a substrate that cathepsin B will specifically recognize and cleave. The probe will be synthesized using organic chemistry, chemical biology, and radiochemistry and its structure will be verified using standard techniques (e.g., NMR and mass spectrometry). Evaluation of its photophysical and pharmacological properties in cells and murine cancer models will follow. Once the probe is assembled, it will be radiolabeled, and injected intravenously into the test subject. The probe will travel to the brain, be chaperoned across the blood-brain barrier, and enter the tumor where cathepsin B will cleave the specific substrate. Once cleaved, the probe self-immolates (disassembles) and fluoresces. The probe also has a radioactive label that can be detected using positron emission tomography. This allows the probe to be later used in humans as its signal can better penetrate the tissues, bones, and organs of the human body. The probe has a modular design meaning the substrate can be exchanged to target a different enzyme of interest. This generalizable strategy is significant and applicable to a variety of human diseases and cancers, especially in the post-Human Genome Project era when hundreds of biomarkers have now been identified.